The overall goal of this proposal is to create a robust one pot, in vitro, multi-gram scale synthesis of activated sugars for drug discovery and biochemical research purposes. Natural product glycosylation is vital for the discovery of new pharmaceutical compounds. While it has been recognized for some time that many biologically active natural products owe much of their therapeutic properties to their glycosylation diversity, methods for creating further diversity for drug discovery have been difficult to develop. One of the challenges is that the modified and rare sugars needed for creating diversity are unavailable or extremely difficult to make, and that the sugars need to be chemically "activated" in order to transfer them to the core natural product molecule. However, new technology has been developed where a chemoenzymatic method is used to attach sugars to natural production aglycons. With this method, a combination of a kinase and nucleotide transferase are needed to form the specific activated sugar and finally a glycosyltransferase is used to transfer the sugar to an appropriate aglycon core. This technology is valuable for drug discovery, as it can be used to produce a library of differentially glycosylated natural product molecules. The Phase II research of this project will continue the improvements in the enzymes needed for this process, including engineering a greater substrate range. The research will then focus on coupling the best enzymes, ATP/NTP regeneration, product recovery, process scale-up and creating novel antibiotic derivatives as a demonstration of the developed technology. PUBLIC HEALTH RELEVANCE: This project is aimed toward developing a method for making a variety of activated sugars for drug discovery purposes. These sugars can used to make derivatives of natural products with new therapeutic properties, for example, antibiotics that are effective against antibiotic-resistant bacteria.